Detection and analysis of poly(amino acids) is important in a variety of commercial and research applications. As used herein, a poly(amino acid) is any homopolymer or heteropolymer of amino acids, including peptides and proteins. Typically, poly(amino acids) are detected and characterized using gel electrophoresis, by solution quantitation assays or by detection on solid supports, such as filter membranes. Small amounts of poly(amino acids) are generally not visible to the naked eye, and must be stained before they can be localized and identified.
Two of the most common methods of staining poly(amino acids) in gels are COOMASSIE Brilliant Blue staining (hereafter referred to as CBB staining) and silver staining. For particular poly(amino acids), silver staining is approximately 100- to 1000-fold more sensitive than CBB staining, but both share some disadvantages. CBB staining and silver staining are time-consuming and yield a narrow range of linear response for densitometric quantitation. Also, the stained gels cannot be blotted for further analysis.
Furthermore, both CBB staining and silver staining are colorimetric--proteins are detected by the presence of colored or opaque bands in the electrophoresis gel. The use of luminescent reagents to detect proteins offers the possibility of greatly enhanced sensitivity and increased linear quantitation range, while simultaneously increasing the ease of use of the staining reagent. By "luminescent" is meant any reagent that is fluorescent, phosphorescent, chemiluminescent, or electroluminescent.
Fluorescent reagents have previously been used for staining poly(amino acids), such as the dye Nile red (9-diethylamino-5H-benzo(.alpha.)phenoxazine-5-one) (see for example Daban et al., ANAL. BIOCHEM. 199, 169 (1991)). Selected styryl and merocyanine dyes have also been utilized as fluorescent stains for poly(amino acids) in gels, on membranes or other supports (U.S. Pat. No. 5,616,502 to Haugland et al., hereby incorporated by reference). While staining with small organic dyes is very rapid, relatively insensitive to poly(amino acid) composition, does not require destaining, and is typically very sensitive, organic fluorescent dyes typically suffer from the drawback of high background noise.
Selected metal complexes exhibit long-lived luminescent emission, and permit "time-resolved" detection at a point after illumination, reducing the interference from short-lifetime fluorescence in the sample to essentially zero.
Luminescent metal chelate stains including europium complexed to bathophenanthroline disulfonate have been described (see M. J. Lim, et al., ANAL. BIOCHEM. 245, 184-195 (1997), and International Publication No. WO 97/20213). The europium-based stain has been shown to be useful for detection of low-nanogram quantities of proteins immobilized on nitrocellulose or polyvinylidene difluoride (PVDF) membranes (patent application Ser. No. 09/080,626, LUMINESCENT PROBES FOR PROTEIN DETECTION, filed May 18, 1998 by Patton et al., now abandoned, incorporated by reference). This method of staining proteins is highly resistant to photobleaching and compatible with popular downstream biochemical characterization procedures including immunoblotting, lectin blotting and mass spectrometry. Disadvantages of the bathophenanthroline disulfonate/europium stain are that the dye can only be visualized using 302 nm UV-B illumination, and exhibits intense blue fluorescence as well as the desired emission maxima of 595 and 615 nm. In addition, the bathophenanthroline disulfonate/europium stain exhibits two emission peaks of roughly the same intensity at 595 and 615 nm, requiring detection across a 24 nm emission window to collect all of the luminescent signal.
Another recently developed luminescent metal chelate stain utilizes ruthenium instead of europium complexes to detect proteins (as described in copending patent application Ser. No. 09/429,739, filed Oct. 27, 1999 by Bhalgat et al., incorporated by reference). The stain offers many of the same advantages as the bathophenanthroline disulfonate/europium stain, but with the additional benefits of higher detection sensitivity, compatibility with most laser-based gel scanners, compatibility with UV epi- and trans-illuminators and minimal cross-reactivity with nucleic acids. However, the ruthenium-based complex exhibits a fairly broad emission peak (100 nm) that may complicate multicolor visualization procedures, and is difficult to remove from bound protein--a significant drawback in some applications.
The europium complexes of the instant invention are roughly 10 times brighter than the bathophenanthroline disulfonate/europium stain described above. Furthermore, the intense blue fluorescence that resulted from the presence of uncomplexed ligand has been eliminated because the compounds of the instant invention are substantially more thermodynamically stable. Proteins stained with the europium complexes of the invention are readily visualized with either UV-A, UV-B, or UV-C illumination, and display a single narrow emission peak. The instant complexes are also easily removed from proteins by increasing the solution pH of the stained sample, making them fully compatible with matrix-assisted laser desorption mass spectrometry, biotin/streptavidin detection systems and immunoblotting.
The europium complexes used to practice the method of the instant invention are highly stable, even in relatively dilute solution, and bind strongly to proteins in solution, on membranes, in biological cells, in moderately acidic solutions, and in electrophoretic gels, yielding bright, long-lifetime, visible luminescence. The complexes exhibit exceptional photostability, allowing long exposure times for maximum sensitivity. The complexes are readily removed from proteins by incubating membranes at mildly alkaline pH. The reversibility of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting, biotin-streptavidin detection and mass spectrometry.
TABLE 1 Comparison of Luminescent Protein Stains: Characteristic: Compound 1 Eu(BPDS).sub.3.sup.3-.dagger. Ru(BP).sub.2 (BPDS).dagger-dbl. Excitation UV-A (360 nm), UV-B (302 nm) UV-B (302 nm), source UV-B (302 nm) Epi-illumination.sup.2 450-532 nm compatibility UV-C (254 nm) Epi- or trans- Epi-illumination.sup.1 illumination Emission Em.sub.max = 615 nm Em.sub.max = 615 nm Em.sub.max = 610 nm source compatibility Reversibility of Readily reversible Readily reversible Fairly permanent staining Emission band- 8 nm 28 nm 100 nm width at (both peaks) half height Stability Stable to dilution Very unstable Stable to dilution Time-resolved T.sub.1/2 = 25-50 .mu.sec T.sub.1/2 = 25-50 .mu.sec T.sub.1/2 = 1-2 .mu.sec fluorescence (theoretical) (theoretical) (theoretical) Interaction with Binds protein Binds protein Binds protein protein Sensitivity for 6 ng/mm.sup.2 47 ng/mm.sup.2 12 ng/mm.sup.2 membrane- bound proteins .dagger.BPDS = bathophenanthroline disulfonic acid .dagger-dbl.BP = bathophenanthroline .sup.1 Excitation maximum is about 355 nm .sup.2 Excitation maximum is about 290 nm